Project Summary Infertility affects 15% of couples who wish to have a child and in about half of these cases the male partner contributes to the inability to conceive. Continuous sperm production by the testis is dependent on the spermatogonial stem cell population (SSC). The SSCs have the capacity to divide to produce more stem cells as well as produce cells that will eventually differentiate into sperm. The maintenance of the SSCs is regulated by a complex regulatory environment called the stem cell niche. The niche is comprised of many factors produced by several testicular cell types including, Sertoli, Leydig, myoid, and macrophages. Furthermore, the stem cells are typically positioned near the vasculature suggesting that circulating factors, such as hormones, also contribute to the SSC niche. How this complex niche manages the SSCs and how the SSCs incorporate the multitude of signals provided by the niche is not understood. To identify and investigate regulatory mechanisms governing SSC maintenance, this study utilizes the genetic and experimental attributes of the zebrafish model. A mutagenesis screen was previously carried out to identify mutations that disrupted germ cell development in adult zebrafish. This is an unbiased approach to interrogate the genetic regulation underlying germ cell development. We hypothesize that mutants with defects in either the SSCs or SSC niche will exhibit a failure in maintenance of the germ line in juvenile/adult stages. Thus, we predict that the affected genes in such mutants will either produce a component of the SSC niche or function in the SSCs for responding to niche signals. This proposal is aimed at the investigation of three new zebrafish mutant lines that exhibit loss of germ cells in juvenile or adult stages in order to gain novel insights into SSC regulation. In Aim1, we will characterize the molecular and cell biological causes of SSC loss in the vacant (vnt) mutant zebrafish. In these mutants, testes appear normal in early adulthood, but germ cells are rapidly depleted leading to eventual germ cell loss and sterility. Two candidate mutations have been found and the proposed experiments will identify the gene leading to this mutant phenotype, determine if the gene functions in the niche or SSCs, and define the defects in SSCs. In Aim2, characterization of two new mutant lines that also exhibit germ cell loss as adults will be carried out. The proposed project will utilize established next generation sequencing techniques to identify the causative mutations, and therefore the affected gene, in each of these mutant lines. In addition, analysis of SSC defects will be performed to confirm that the SSC population is specifically affected in these mutants. This approach will lead to the identification of genes that have not been previously implicated in SSC maintenance and to future studies on novel mechanisms underlying this process. Knowledge gained from this study can be applied to a better understanding of human reproductive health and to improved methods for diagnosis and treatment of infertility, assisted reproductive technologies, and development of male-targeted family planning methods.